The present invention relates generally to osmometry and, more particularly, to measuring the osmotic pressure of complex solutions including, but not limited to, emulsions, dispersions, and charged and uncharged polymer solutions.
A variety of methods and devices are known for measuring the osmotic pressure exerted by solvent molecules diffusing through a semipermeable membrane. Commercially available devices for measuring the osmolality of solutions via membrane osmometry include the 4400 series colloid osmometers made by WesCor (Logan, Utah) and the type 1.00 Knaur membrane osmometer. Other devices for measuring the osmolality of solutions through vapor pressure osmometry are commercially available. An example of this type of device is VPO model 070 made by UIC Inc. (Joliet, Ill.). Still another known osmometer device operates by measuring freezing point depression. An example of this type of device is the Precision Systems Inc. (Natick, Mass.) Osmette XL product line. Other known osmometer variants include isopiestic vapor equilibrium osmometers and submerged dialysis bag osmometers. In addition, osmolality has been determined by measuring boiling point elevation. The vapor pressure osmometer, boiling point elevation, freezing point depression, and isopiestic methods measure the oncotic pressure (the osmotic pressure exerted by colloid in the solution) of the solution, that is, the osmolality of the solution including the contribution of low molecular weight components such as salts. The present invention is directed to methods for measuring the osmotic pressure of solutions, excluding the contribution of small molecules.
Membrane osmometers and the dialysis bag techniques measure the solution equilibration across a semi-permeable membrane, thus excluding the direct contribution of small permeable molecules. One example of a membrane osmometer is taught in U.S. Pat. No. 4,150,564, titled xe2x80x9cOSMOMETER FOR COLLOID OSMOMETRY,xe2x80x9d by Wayne K. Barlow, et al., Apr. 24, 1979. The present invention is an improvement upon existing membrane osmometer designs, but relies on the same basic principle of establishing an equilibrium across a semi-permeable membrane. Typical membrane osmometers employ pressure transducer technology to directly measure the evolved osmotic pressure difference between a reference cell and the sample solution across a semi-permeable membrane. In the present invention, transducer technology is not required for measuring the osmotic pressure. Typical commercial osmometers are designed to minimize the sample volumes by introducing the sample into a meandering channel above the semipermeable membrane that maximizes surface contact while minimizing sample volume. This meandering channel geometry is eliminated in the present invention because it limits the usefulness of commercial osmometers to low viscosity, non-fouling solutions and is particularly unsuited for complex solutions (dispersions and emulsions).
Dialysis bag techniques, (Essafi, W. Structure Des Polyelectrolytes Fortement Charges, PhD thesis, Universite Pierre et Marie Curie, Paris, 1996) involve filling a semi-permeable dialysis bag with the sample solutions of unknown osmotic pressure and immersing it in a large volume of solution of known osmotic pressure. The sample changes concentration until an osmotic equilibrium is established. Then the sample is removed and its concentration at that known osmotic pressure is determined using other techniques (spectrophotometrically or gravimetrically). The present invention may be used in a mode of operation similar to this. In this mode of operation the sample solution is allowed to equilibrate to a known imposed air pressure. Then the concentrated sample is removed and the concentration determined separately. However, this method of operation is not the optimal nor is it the preferred embodiment of the method of the present invention.
The prior art also suggests use of polymer solutions of known osmotic pressure to have as reference solutions (rather than solvent) using commercial osmometers or other direct force measurement techniques to allow for measurement of higher osmotic pressure solutions, (Rau, Donald C.; Parsegian, V. Adrian. Direct Measurement Of Temperature-Dependent Solvation Forces Between DNA Double Helixes. Biophys. J. (1992), 61(1), 260-71; Sidorova, Nina Y.; Rau, Donald C. Removing Water From An EcoRI-noncognate DNA Complex With Osmotic Stress. J. Biomol. Struct. Dyn. (1999), 17 (1), 19-31). This method works reasonably well for extending the pressure range available on commercial osmometers but does not properly treat the Donnan equilibrium established for charged species thereby potentially leading to erroneous results with charged polymers. This method of extending the pressure range is not needed in the operation of the present invention as the reference solution is simply dialyzate.
The present invention capitalizes upon advances in stirred cell dialysis chamber technology to improve osmometer design. Stirred cell dialysis chambers are available from, for example, Amicon, (Beverly, Mass.). The principal use of the dialysis chamber in the prior art is to either concentrate a sample solution or to remove small molecule impurities by exhaustive flushing with pure solvent. The stirred cell is designed to be dismantled easily for cleaning. It has a magnetic stirring rod suspended above the membrane to keep the solution well stirred and to sweep clean the surface of the membrane. It can withstand more than 75psi of external pressure, far exceeding the pressure measurable in conventional membrane osmometers (xcx9c1-3 psi).
It is therefore an object of the present invention to provide a method for determining the concentration of a solution having a predetermined osmotic pressure and/or generating a solution with a predetermined osmotic pressure.
It is a further object of the present invention to provide a method for determining the concentration of a solution having a predetermined osmotic pressure and/or generating a solution with a predetermined osmotic pressure which allows for easy dismantling and cleaning so that complex fluids (the emulsions, dispersions, etc.) which would foul traditional meandering channel membrane osmometers can be measured.
A further object of the present invention is to provide a method for determining the concentration of a solution having a predetermined osmotic pressure and/or generating a solution with a predetermined osmotic pressure which includes stirring the surface of the membrane continually thereby reducing surface fouling and removing bubbles that hamper the accuracy and reliability of prior art osmometers when used to measure complex fluids.
Yet another object of the present invention is to provide an osmometer which keeps the sample well mixed thereby avoiding particle settling and the development of surface concentration gradients.
It is a further object of the present invention to provide a method for determining the concentration of a solution having a predetermined osmotic pressure and/or generating a solution with a predetermined osmotic pressure.
Still another object of the present invention is to provide a method for determining the concentration of a solution having a predetermined osmotic pressure and/or generating a solution with a predetermined osmotic pressure which can be used for measuring osmotic pressure of high viscosity solutions (above about 100 cp).
The foregoing and numerous other features, objects and advantages of the present invention will become readily apparent upon reviewing the detailed description, claims and drawings set forth herein. These features, objects and advantages are accomplished by providing a sample cell with a removable pressurizing lid through which a sample solution may be introduced into the chamber of the sample cell. The sample is introduced above a membrane residing in the sample cell with a meandering dialyzate cell positioned below the membrane. Pressurized gas is introduced into the sample cell via a pressure regulator. Once the sample is forced through the membrane and dialyzate begins to emerge through a transparent dialyzate tube from the meandering dialyzate cell, the pressure through the pressure regulator is reduced until flow ceases. A magnetic stirrer is used to continually sweep the surface of the membrane. The primary purposes of the meandering dialyzate cell are to provide a structure for collecting dialyzate with maximum surface area contact with the membrane (for fast equilibration with the sample cell) which minimizes the volume of dialyzate relative to the volume of sample in the sample cell. However, those skilled in the art will recognize that the present invention can be practiced without a meandering dialyzate cell. Instead, the sample cell can easily be provided with a support structure other than a meandering dialyzate cell to support the membrane therein. Similarly, the sample cell can also be provided with a different dialyzate collector (e.g. a funnel). The support structure for the membrane and the dialyzate collector do not have to be integrated as is the case with a meandering dialyzate cell. However, these alternative arrangements would likely (although not necessarily) increase the volume of dialyzate needed before a meniscus is achieved in the transparent dialyzate exit tube.
The use of a pressure regulator and an accurate pressure gauge allow for precise control and measurement of the applied air pressure on the sample. By systematically varying the applied air pressure until the visually observed liquid meniscus of the dialyzate in the dialyzate tube is stationary, the osmotic pressure of the solution across the dialysis membrane is exactly balanced by the imposed air pressure. Further, the method and apparatus of the present invention allow for the progressive adding of known amounts of diluent to the sample solution and mixing the new dilutions directly in the sample cell. This is achieved practically by simply placing the entire cell on an accurate balance and weighing the amount of added solution before each osmotic measurement. Calibration of the apparatus of the present invention does not require equilibration of a sample. Rather, calibration is a simple and fast procedure based on accurately measuring the air-line pressure. This is in marked contrast to the difficulty of repeatedly calibrating transducers and properly seating membranes in prior art membrane osmometers. This leads to a significant advantage in the amount of time required to operate the apparatus.
The preferred method of the present invention for determining the concentration of a solution having a predetermined osmotic pressure and/or for generating a solution with a predetermined osmotic pressure starts from a concentrated sample with a higher osmotic pressure than the desired predetermined osmotic pressure. A second method is given below for sample solutions which need to be concentrated to increase the osmotic pressure to the desired predetermined osmotic pressure. The basic idea is to measure the osmotic pressure of a series of gradual solvent dilutions of the concentrated sample, performed in the sample cell, until a final sample solution with the desired osmotic pressure is achieved. This is done by first placing in the sample cell a more concentrated sample with higher osmotic pressure than the desired predetermined osmotic pressure. The sample cell 12, of course, includes a membrane 16 supported therein. The sample is then sealed in the sample cell 12 and gas is then introduced at a predetermined elevated pressure. The osmotic pressure of the sample can then be determined by varying the applied gas pressure until the dialyzate meniscus is stationary. Next the sample cell 12 is placed upon a balance and a known weight of diluent is added directly to the sample cell 12 so as to dilute the sample 64 to a concentration calculated to be slightly more than the desired osmotic pressure. A simple linear method of estimation of the amount of diluent to add based upon equating the ratios of concentrations to the ratio of measured and desired osmotic pressure will typically suffice. The diluted sample is resealed and the osmotic pressure is again measured. This process is repeated until the osmotic pressure desired is achieved. The known dilutions allows for the calculation of the final concentration from the initial concentration of the concentrated sample. This is the fastest procedure.
If the added diluent ever lowers the osmotic pressure of the sample below the desired osmotic pressure then the sample 64 is placed in the sample cell 12 including therein a membrane 16 supported therein. Then the remaining steps of the method are: sealing the sample 64 in the sample cell 12; introducing gas at a predetermined elevated pressure substantially equal to the predetermined osmotic pressure; and generating a dialyzate by allowing the solution to dialyze through the membrane 16 until equilibrium is achieved and no more dialyzate passes through the membrane 16 while the sample cell 12 is maintained at the predetermined elevated pressure, the sample remaining in the sample cell 12 having an osmotic pressure substantially equal to the predetermined elevated pressure; collecting the dialyzate of said generating step; and weighing the dialyzate of said collecting step to determine the concentration of the solution remaining in the sample cell. Even when a slight over dilution occurs the process is reasonably fast because the over diluted sample has an osmotic pressure close to the desired osmotic pressure. This is important since using dialysis to concentrate a sample can be quite a time consuming process compared to the fast measurement process just described.
If the sample available has a lower osmotic pressure than desired (a lower concentration) then the following more time consuming method must be used. This alternative method of the present invention for determining the concentration of a solution having a predetermined osmotic pressure and/or for generating a solution with a predetermined osmotic pressure is performed by placing a sample 64 of a solution which has a lower osmotic pressure than the desired predetermined osmotic pressure in the sample cell. The sample is placed into the sample cell 12, the sample cell 12 including therein a membrane 16 supported therein; sealing the sample 64 in the sample cell 12; introducing gas at a predetermined elevated pressure substantially equal to the predetermined osmotic pressure; and generating a dialyzate by allowing the solution to dialyze through the membrane 16 until equilibrium is achieved and substantially no more dialyzate passes through the membrane 16 while the sample cell 12 is maintained at the predetermined elevated pressure, the sample 64 remaining in the sample cell 12 having an osmotic pressure substantially equal to the predetermined elevated pressure; collecting the dialyzate of the generating step; and weighing the dialyzate of the collecting step to determine the concentration of the solution remaining in the sample cell 12. This is a more time consuming method because the sample must dialyze to a higher concentration. It is unavoidable, however, if the sample 64 needs to be concentrated.
Generating a solution with a predetermined osmotic pressure was simply not possible with prior art osmometers. The methods of the present invention to generate such a solution and to determine its concentration are faster than even the mere measurement of the osmotic pressure of an unknown sample using prior art osmometers.
For improved accuracy the height of both the dialyzate liquid column in the dialyzate tube and the sample reservoir are recorded and used to correct for the known hydrostatic pressure difference. The entire apparatus is placed on a hot plate for temperature control. This method is very fast, approximately 4-50 times faster than other methods. Because of the speed of measurement of the present invention, the osmotic pressure of chemical reactions can be monitored as a function of time or temperature in the sample cell. This feature potentially opens new areas of research for instance in medicine, or polymer chemistry where the appearance or disappearance of reactants is accompanied by an osmotic pressure change.